Berries and leaves from six varieties of Carpathians’ sea buckthorn (Hippophae rhamnoides L., ssp. Carpatica) were analysed for their carotenoid composition (free and esterified) using a combination of HPLC-PAD, GC–MS and UHPLC–PAD–ESI-MS techniques. GC–MS techniques revealed the fatty acid profile specific for each berry variety, while targeted UHPLC–MS analysis identified the fatty acids involved in carotenoids esterification: palmitic (C16:0), myristic (C14:0) and stearic (C18:0). Total carotenoid content varied between 53 and 97 mg/100 g dry weight in berries, and between 3.5 and 4.2 mg/100 g DW in leaves. The carotenoid di-esters represented the main fraction among berry varieties having zeaxanthin di-palmitate as major compound, while leaves contained only free carotenoids like lutein, ß-carotene, violaxanthin and neoxanthin. Principal component analysis identified the suitable carotenoid biomarkers characteristic for the Carpathians’ sea buckthorn from Romania with contribution to their taxonomic classification and authenticity recognition.